“Working at Bioline as Distributor Marketing Manager involves travel all over Europe, Scandinavia and occasionally as far as California and Dubai, meeting many interesting people. A rewarding part of the role is becoming an extended part of our distributors business; discovering and planning how to assist with development and growth through the sales of Bioline product families, this has also allowed me to increase my knowledge of international sales, a wide range of cultures, molecular biology and the life science industry.

Operating so closely with distribution partners and our direct sales and marketing counterparts globally has given me a wealth of business development and marketing knowledge, following trends in technology, sales and markets globally. It is good to know that being in this industry contributes to strides forwards in terms of how we combat, diagnose and prevent disease, advance agricultural and environmental research and improve forensic techniques.”

Epigenetics can be thought of in broad terms as the range of phenomena that bring phenotype into being without changing the underlying DNA sequence. Consider that the vast majority of cells within multicellular organisms have identical genotypes yet exhibit distinct cellular functions with different gene expression profiles that are stable and, in many instances, heritable. Thus the epigenetic landscape of an organism is an important function of cellular differentiation and much remains to be learned about these processes. Bioline offers a suite of molecular tools, proteomics reagents, assay panels, and complete assay design services to assist research efforts in the field of epigenetics.

Epigenetics – dynamic and global regulatory mechanisms
Our genome is not in a static state, and although our genotype strongly influences resulting phenotype, there are many differences in gene function and expression that cannot be attributed to changes or differences in DNA sequence alone. A range of epigenetic processes have been identified that post-synthetically modify DNA or interact with the proteins intimately associated with DNA, controlling the journey of transcription and ultimate translation of protein to subsequent phenotype. These mechanisms include a range of histone modifications that affect chromatin structure, subsequent DNA packing and chromosome formation, thus controlling the availability of genes to transcription mechanisms.

Methylation patterns of DNA have also been identified as strong drivers of gene activation/inactivation. Nearly all DNA methylation in mammalian genomes occur at cytosine residues of CpG dinucleotides, and high density CpG regions (or islands) that are methylated correlate with transcriptional repression. DNA methylation has been shown to play a role in important cellular processes including X chromosome inactivation, plus changes in methylated state have been identified in diseases such as cancer. DNA methylation patterns can change in response to environmental factors such as diet or exposure to toxins, and it has been shown that some of these adaptive changes are passed on to future generations. There are a range of methods to detect and quantify DNA methylation including PCR, Real-Time PCR, or High Resolution Melt (HRM) analysis, and Bioline provides reagents and kits to support research in these areas.

The role of RNA, in particular non-coding RNA (ncRNA), has been established as a major controlling element of multiple epigenetic events. In addition to the established functional RNAs (mRNA, tRNA, and rRNA) involved with processing of the coding RNA, and associated small nuclear and nucleolar (snRNA and snoRNA) RNAs that splice and modify RNA nucleotides, other small ncRNAs such as microRNAs (miRNA) and short interfering RNA (siRNA) have been identified in epigenetic mechanisms - involved in regulation of target mRNA and chromatin. Long non-coding RNAs have also been implicated in gene regulation, thus there is a complete network of ncRNAs intimately involved in epigenetic processes. These elements can be studied through deep genome sequencing approaches, as well as targeted PCR or Real-Time PCR assays to detect, identify, or quantify the various ncRNA elements, and Bioline offers a range of molecular tools for these analyses.

Epigenetics – the role of miRNAs
There are over 2,000 different short, single stranded RNA molecules currently identified as unique human mature miRNAs in the miRBase sequence database. After binding with the RNA-induced silencing complex (RISC), these miRNA complexes interact with target mRNA to induce degradation or cleavage, or to block translation – thus miRNAs are key epigenetic regulators of post-transcriptional gene expression. With involvement in many key cellular functions such differentiation, proliferation and apoptosis, the study of miRNAs and their mechanisms is key to understanding normal cellular development as well as aberrant disease states.

In addition to the full range of physiological and pathophysiological functions regulated by miRNA activity, they have also shown to be important regulators of other epigenetic mechanisms such as DNA methyltransferases and histone deacetylases. Conversely, DNA methylation and histone modification have been linked to regulation of expression of certain miRNAs, and there is also evidence of miRNAs regulating other miRNAs – thus an epigenetics-miRNA regulatory circuit exists, creating a complex and intricate gene expression process involving feedback mechanisms and elements of self-regulation. The regulatory landscape of miRNAs is far from straight forward and much remains to be understood regarding the expression of miRNAs and their subsequent regulatory roles. In addition to individual molecular tools and kits for isolation, amplification and identification of miRNA targets, Bioline in conjunction with MiRXES, a Singapore-based life science research company, have developed the EPIKTM range of miRNA assay panels representing key miRNA targets identified using extensive bioinformatics mega-studies and linked to important disease states such as cancer.

Epigenetics – Applications and Challenges
With epigenetic drivers involved in so many cellular processes, there are any number of applications in which to study the various mechanisms of DNA methylation, histone modification or non-coding RNA control of gene expression. Whether you’re sequencing an entire genome, isolating nucleic acids, amplifying target sequences or studying the resulting proteins, Bioline has the individual reagent, complete kit, individually designed assay service, or ready to use panel to support all of your research efforts.

Epigenetics and cell differentiation mechanisms
Understanding how a single cell grows into a complete multicellular organism still remains somewhat of a puzzle, how are the tissue specific patterns of gene expression established and maintained throughout the life of an organism? How do cells interpret signalling cues, and how do stem cells both maintain their stem cell properties and differentiate progeny into various cell lineages? Given that the majority of cells within this system contain genetically identical material it is clear that epigenetic factors play a key role during organism development, as well as in stem cell renewal and differentiation throughout life. There is still much to be understood about the drivers behind embryogenesis, as well as the signals that fine-tune cell functions and gene expression changes throughout the lifespan of an organism. Although key genes and regulators of those genes have been identified in these processes, any number of epigenetic factors are involved, simultaneously interacting with each other and the local and external environment to bring about the expression of the unique set of genes that define each and every one of us.

Understanding epigenetic inheritance
Many epigenetic changes within cells act as one-way barriers, ensuring that cell-type specification and activity is stable and maintained, thus there are clear examples for mitotic inheritance of epigenetic regulation. Mechanisms exist for propagation of DNA methylation patterns, however little is known about how epigenetic flexibility is maintained during cell development and differentiation, and non-DNA based epigenetic inheritance is still poorly understood. Meiotic inheritance is even more controversial. During mammalian sexual reproduction, the epigenome must be reset back to a totipotent state in preparation for the development of the next generation. Given this erasure of somatic epigenetic signatures, it is difficult to understand how epigenetic changes could be transgenerationally, or meiotically inheritable. Intriguing multi-generational studies have reportedly identified specific epigenetic markers and associated phenotypic characteristics in offspring linked to environmental exposures such as diet restriction or toxin exposure in grand-parents, suggesting transgenerational epigenetic effects may indeed exist. However many studies in this area have been contested, and a mechanism for such inheritance has not yet been defined. There is certainly a wide gap in our knowledge and research efforts persist with mixture of excitement and caution.

Epigenetics in cancer and disease states
Given their key role in so many important cellular functions it stands to reason that aberrant epigenetic processes have been identified in many cancers as well as other disease states. Hyper-methylation of CpG islands has been identified in a range of cancers resulting in subsequent silencing of tumour suppressor genes, DNA repair genes or other processes important to normal cell growth. Epigenetic factors have also been linked to inherited disorders associated with mental retardation such as Fragile X syndrome, Prader-Willi, and Rett syndrome. Epigenetic changes have been observed in response to external environmental factors, and as such, epigenetic links are being investigated for conditions such as obesity, diabetes or heart disease. Although there are many clear correlations between epigenetic states and disease, there are many more confounding studies and results that have been difficult to replicate. There is vast variation in epigenetic alterations across cell populations within an individual, let alone across populations, meaning that many epigenetic studies of disease bring up more questions than they answer. In addition, it is often difficult to determine whether epigenetic changes are a cause or consequence of disease state. Nevertheless, epigenetic profiling continues, and epigenetic therapeutics are also being developed, as efforts continue towards a greater understanding of these important regulatory elements and their roles in disease.

Thank you Tiffany Kosch from James Cook University for your review of SensiFAST™ Probe No-ROX Kit

"I performed a comparison of of Bioline's SensiFAST™ Probe No-ROX Kit (cat. BIO-86005) against several other qPCR master mixes. The Bioline product performed the best overall (i.e. better efficiency and sensitivity, lower cost). I have attached a graph of the Quantitation analysis from my trial run of this product. I have also been very impressed with Bioline customer service and really appreciate that they are willing to send free samples of most of their products."

The path from discovery through development to commercial drug is a long and costly one. Estimates from the big pharmaceutical companies range between $1.2 - 1.8 billion per new prescription drug approval, taking into consideration the many targets that fail somewhere along the lengthy process, although some smaller companies have been successful with much smaller investments in the vicinity of $500 million. Regardless of the overall cost, it is a significant investment, with the average time to launch a successful drug around 12-15 years. Of the thousands of compounds that manage to reach Phase I clinical trials and continue through the development process, barely 10% will ultimately gain approval for use, thus the process is far from efficient, and these figures do not even take into account all the work done in the discovery phase of the process.
There is clear value, both economic and social, in the successful launch of a new therapeutic and research and development efforts persist across the board to continue to find new therapeutics to support the growing number of cancer patients, neurobiological diseases, heart disease along with all the other ailments that threaten our health. Bioline have a full range of products to support the drug development and discovery process from molecular screening for disease characterization through to cloning for functionality tests.

The Phases of Early Drug Discovery
There are universally accepted stages or phases to the typical drug discovery and development cycle. The process of early discovery mainly focuses on characterizing and understanding a disease and associated mechanisms, this work often occurs in clinical research or academic settings. Whereas the process of identifying potential drug candidates can take place in the traditional large scale setting of a pharmaceutical laboratory or, as is happening more often, in smaller, independent laboratories who form commercial entities to take advantage of a potentially viable candidate.

Bioline offers a range of products from individual reagents and targeted kits, right through to large scale assay panels and fully validated assay design services that lead to better understanding of a specific disease pathway, help to characterise a model organism or interrogate a tissue sample. High throughput sequencing products aid in screening and canvassing whole genomes or disease states, while cloning reagents assist with functional investigations. ISO13485 manufacturing supports transition from pre-clinical to clinical trial phases, offering the standard and quality required for strict regulation purposes.

The initial research phase of drug discovery involves understanding an essential mechanism of the disease in question and forming a hypothesis that inhibition or activation of an element within that mechanism will result in a therapeutic outcome in disease state. From here, targets are identified that are both measurable and accessible to a potential drug molecule. Targets can be in the form of proteins, genes or RNA, and large scale screening and data mining is often employed to assist in this phase of the process. Once a suitable target is identified it undergoes a thorough validation process. Techniques such as gene knockouts and the use of transgenic animal models allow researchers to study the role of the investigated target within the disease pathway in an attempt to fully understand the effects of manipulation of the target on downstream events. This is important not only in terms of eventual efficacy of the future drug, but also in terms of safety, both key factors that directly impact the likelihood that the drug development process will be successful.

The next phase of discovery focuses on identifying compounds that will interact with the target molecule. Known as the hit identification and lead discovery phase this usually involves high throughput screening of millions of bioactive compounds, using as much automation as possible to streamline the testing process, as well as virtual screens or compound modelling to help narrow down the candidates. The hit identification and lead generation process is a complex phase and requires the workup of specific functionality assays that will allow for accurate measurement of molecule interaction. Compound screens identify potential hits and chemistry programs aim to tweak the interaction to improve specificity, potency or other physiochemical properties of the hit molecules. Once the field of potential hits has narrowed, more specific tissue-based screens can look at in vivo effects of target interaction and ensure that candidates can operate within an intact system. The lead generation work is designed to refine the hit series and, through investigating compound structure and activity, maximize the potency and specificity of successful candidates. Candidates are then run through pharmacokinetic/pharmacodynamics studies, usually in animal models, in order to understand dose linearity and metabolic profiling. Successful lead compounds are then transferred to the drug development phase.

Drug Discovery to Development – Challenges and Costs
There are a multitude of barriers and difficulties that contribute to the minimal success rate within the drug development process, starting with our level of knowledge and understanding of disease states, particularly complex diseases that involve a number of genetic elements or biological phases and pathways – such as cancer. Many investigators have identified the need to better understand disease mechanisms before effective therapeutics can be developed, and those within the academic and clinical research field are often best placed to provide this important data. Moreover, identifying and understanding the phenotypic heterogeneity that exists within a patient population can often be key to the success or failure of a drug candidate. Examples exist where approval of drug usage is dependent on upfront genetic screening in order to stratify patient populations.

Another challenge surrounds the use of biomarkers – surrogate or indirect indicators of a patient status or disease state that are often used to measure the effectiveness of a drug or treatment regime. Extensive and costly clinical trials are required to ensure a chosen biomarker can reliably predict clinical efficacy across a patient population and that the measured outcomes are meaningful and reproducible. Reproducibility is in fact a major concern throughout the drug development process, calling for the use of effective and representative modelling systems and activity assays. Although animal models are often employed, results from these systems do not always easily translate to human biology thus, stem cell-based models that more accurately replicate the disease microenvironment are gaining favor in this field.

Technologies and Approaches to Drug Discovery
There are a number of different approaches to drug discovery aimed at maximising the overall efficiency of the process. The traditional mainstay of drug discovery was housed in the phenotypic approach – where the effects of test compounds on tissues, cells or whole organisms was monitored to see how that molecule modified the disease phenotype. The issue with this approach was that often the mechanism of interaction was not fully understood and there was potential for multiple targets of the test compound, which often led to side-effects in clinical trials downstream. The advent of deep sequencing and the genomics era has been hailed with great expectations to better highlight targets and pathways amenable to drug discovery, as well as improve patient stratification and enhance clinical development programs. Now, as the cost of sequencing continues to drop, this technology not only becomes more accessible for use during discovery, it is also being identified for use as a companion diagnostic to target patients that will more likely benefit from treatment. Bioline offers a range of sequence preparation and support products for both Sanger and next generation sequencing projects to assist both the drug discovery process and development of potential diagnostics.

The systems biology approach to drug discovery and development capitalises on bioinformatics developments and improvements in computing power, incorporating information form genomic sequencing, biological function databases, microarray data and gene expression databases, and complex calculations of kinetics and molecular interactions, to create computational models within which to test a vast array of targets and candidate molecules. This approach takes advantage of the vast amount of research already done and attempts to make sense of the mountains of data – identifying enriched gene sets or pathway maps, classifying diseases or stratifying patients based on gene expression profiles, or attempting to understand the observed changes in gene signatures of disease states through a range of interrogation or reverse causal reasoning hypotheses. Once candidates are identified in this virtual model, they can be taken through the various practical stages for testing.